Biodeterioration of Some Cultural Heritage in Isfahan City, Iran

Nature Environment and Pollution Technology ISSN: 0972-6268 Vol. 13 No. 1 pp. 141-146 2014 An International Quarterly Scientific Journal Original Research Paper Biodeterioration of Some Cultural Heritage in Isfahan City, Iran

H. Modaresi, N. Bahador and M. Baserisalehi* Department of Microbiology, Science and Research Branch, Islamic Azad University, Fars, Iran *Department of Microbiology, Kazeroun Branch, Islamic Azad University, Kazeroun, Iran

ABSTRACT Nat. Env. & Poll. Tech. Website: www.neptjournal.com The aim of this study was to isolate and phenotypical identification of microorganisms involving in biodeterioration of four historical places in Isfahan, Iran. For this purpose two hundred and ten samples Received: 2-7-2013 were taken from the stones of Chehel Sotoun, Hasht Behesht, Alighapou and Imam mosque. For bacterial Accepted: 12-8-2013 isolation blood agar and modified Bristol medium were used and for screening of fungal isolates potato Key Words: dextrose agar was used. Then the samples were serially diluted and poured on the selected media. For Biodeterioration bacterial isolates the plates were incubated at 30°C for 48 hrs and for fungal isolation the plates were kept at Historical places room temperature for 4 weeks. The pure colonies were identified using phenotypical and molecular Isfahan identification. Fungi were characterized using macroscopic characteristics and microscopic arrangements. Molecular technique The results obtained from this study indicated that the dominant isolated bacteria were Bacillus licheniformis, Bacillus thuringiensis and Halomonas sp. In addition the results showed that the fungal isolates belonged to Genus: Alternaria, Cladosporium, Penicillium, Absidia and Shaccharomyces. Furthermore, the most polluted area was Chehel Sotoun which is located in the traffic part of the city. Overall, stone materials of cultural heritage are constantly exposed to biodegradation by microorganisms and presence of organisms including algae, fungi and bacteria are likely to affect or even cause physico-chemical changes.

INTRODUCTION biodeterioration. Because of photosynthetic nature, the first coloniser of historical places are algae and cyanobacteria; There are many organisms which are involved in these microorganisms can deteriorate stone either chemically biodeterioration like bacteria, fungi, archaea, algae, and li- or mechanically. On the other hand, lichens, which are re- chens. Presence of these organisms and different types of sistant to desiccation and extreme temperatures, are also fre- stones could help the interaction and then make visual and quently associated with biodeterioration of stones (Herrera structural damages in historical places (Gadd 2007). Micro- et al. 2004). Hence, The aim of this study was evaluation organisms within the stones may grow in cracks and pores and characterization of the bacteria and fungi incorporated with different mechanisms (Mc Namara et al. 2006). Indeed, in biodeterioration of historical places in Isfahan city, Iran. biodeterioration may be defined as any undesirable change in the properties of a material caused by the vital activities MATERIALS AND METHODS of living organisms. In recent years biodeterioration of stone monuments and buildings is a well recognized and studied The Isfahan City: Isfahan, historically also rendered in Eng- worldwide (Warscheid 2003, Gordon & Dorn 2005, Nugari lish as Ispahan, Sepahan or Hispahan, is the capital of Isfahan et al. 2009). In some tropical regions with high tempera- Province in Iran, and located about 340 km south of Tehran. tures, relative humidity and heavy rainfall this phenomenon Isfahan is located on the main north-south and east-west favours the growth of wide variety of living organisms (Gadd routes crossing Iran, and was once one of the largest cities in 2007). In other words, along with vital activities the initial the world. The city is famous for its Islamic architecture, deterioration of stone exposed to the outdoor environment with many beautiful boulevards, covered bridges, palaces, and action of agents such as rain, wind, sunlight, and pollu- mosques, and minarets. The Naghsh-e Jahan Square in Isfahan tion may play an important role for physical and chemical is one of the largest city squares in the world and an out- processes. When the surface of the monument has under- standing example of Iranian and Islamic architecture, which gone this process of alteration, living organisms begin to has been designated by UNESCO as a World Heritage Site. colonize the area form biofilms and finally cause structural The city also has a wide variety of historic monuments and damage (Nuhoglu et al. 2006). Although biofilm formation is known for the paintings and history. The city is situated at has an important role in biodeterioration, chemical reactions 1,590 metres (5,217 ft) above sea level on the eastern side of with the material, physical penetration into the substrate and the Zagros Mountains and has an arid climate. Despite its production of pigments are the other main factors in altitude, Isfahan remains very hot during the summer with 142 H. Modaresi et al. maxima typically around 36°C or more (Assari & Mahesh March in Isfahan to prevent contamination from the soil and 2011). ground. Sterile devices were used in the sampling and all of Sampling sites: The historical places of Isfahan are well- the procedures were done aseptically (Nuhoglu et al. 2006). known examples of various civilizations. The four main his- The microbiological studies were carried out under sterile torical places identified in Isfahan were chosen for deter- conditions. mining the biodeteriorative effects of the microorganisms Totally 210 specimens were taken from the stone sur- on stone decay. These places were restored by the Ministry face of all the monuments in January and March 2011-2012. of Culture and Tourism of Islamic Republic of Iran. The char- During the sampling procedures the daytime and night time acteristics of the historical buildings are given below: average temperature was 20 and -2.5°C in January, and 27 Imam Mosque: Formerly known as Shah Mosque is a and 9.3°C in March. The first specimen was taken by means mosque in Isfahan, Iran. The mosque is standing in south of scraping from the stone surface. These samples were used side of Naghsh-i Jahan Square. The mosque was built dur- for microbiological incubating and counting. One gram of ing the Safavid period. It is an excellent example of Islamic each sample was squashed with a mortar and weighed. Then, architecture and regarded as one of the masterpieces of Per- they were put into a 50 mL flask with 10 mL sterile physi- sian architecture. The Imam Mosque is registered, along with ological solution (9g/L NaCl), mixed for 15 min and then -1 -7 the Naghsh-i Jahan Square, as a UNESCO World Heritage serially diluted (10 -10 ) and plated on different microbio- Site. Its construction began in 1611, and its splendor is mainly logical media including: Blood agar and MBM medium for due to the beauty of its seven-colour mosaic tiles and chemolithotrophic bacteria (Vero et al. 1975, Greenberg et calligraphic inscriptions. al. 1985). Preliminary identification of bacteria was based on phenotypical characteristics and then the isolates were Âlî Qâpû: It is a grand palace in Isfahan, Iran. It is located identified using molecular methods. on the western side of the Naqsh-e Jahan Square opposite to Sheikh lotf allah mosque, and had been originally designed Isolation and identification of fungi: For isolation of the as a vast portal. It is forty-eight meters high and there are fungi the samples were cultured on sabouraud dextrose agar seven floors, each accessible by a difficult spiral staircase. containing peptone (10 g), glucose (20 g) and agar (15 g). In the sixth floor music room, deep circular niches are found The medium was supplemented with chloramphenicol and in the walls, having not only aesthetic value, but also acous- cycloheximide (50 and 500 mg/dl). The cultures were incu- tic. The name Âlî Qâpû, from Arabic Âlî, “Imperial or Great”, bated at 26°C and examined twice a week for a total dura- and Turkich Qâpû meaning “gate”, was given to this place tion of 4 weeks. After that, the isolates were examined mac- as it was right at the entrance to the Safavid palaces which roscopically, and microscopically following staining with stretched from the Maidan Naqsh-i-Jahan to the Chahâr Bâgh lactophenol cotton blue. The identification of yeast was based Boulevard. on their microscopic characteristics as a result of germ tube tests and biochemical tests and the identification of moulds Chehel Sotoun: It is a pavilion in the middle of a park at the was based on their macroscopic characteristics (growth pe- far end of a long pool, in Isfahan, Iran, built by Shah Abbas riod, colony morphology, production of pigment on the back II to be used for his entertainment and receptions. The name, of the colony), microscopic arrangements (characteristics meaning “Forty Columns” in Persian, was inspired by the hyphae formation, types of conidia, sizes and shapes of the twenty slender wooden columns supporting the entrance sterigmata, hyphae and organs of reproduction) (Nuhoglu et pavilion, which, when reflected in the waters of the foun- al. 2006). tain, are said to appear to be forty. The palace contains many frescoes and paintings on ceramic. Many of the ceramic pan- Identification of bacteria using PCR technique: For mo- els have been dispersed and are now in the possession of lecular identification single colonies were picked up from major museums in the west. solid medium and subjected to release DNA. DNA extrac- tion was carried out using DNA PCR kit (Roche-Germany). Hasht Behesht: Meaning “Eight Paradises” is a Safavid era Then the purity of the extracted DNA was assessed by ab- palace in Isfahan. It was built in 1669 and is today protected sorbance at 260 and 280 nm. The extracted DNA with ratio by Iran’s Cultural Heritage Organization (Assari & Mahesh (260/280 nm) of 1.9 was used for PCR. Amplification of 2011, 2012). 16SrRNA gene was performed using Forward and Reverse Isolation and identification of bacterial isolates: The stone primers with sequence of 5´AGGAGGTGACCAAC- samples were taken from the external walls of the monu- CGCA3´ and 5´AACTGGAGGAAGGTGGGGA 3´ respec- ments from 3 m height of four historical buildings. They tively. The PCR products were run on 1.5% (w/v) agarose were put into the sterile screw capped tube in January and gel. PCR products were electrophoresed at 75 V for 20 min

Vol. 13, No. 1, 2014  Nature Environment and Pollution Technology BIODETERIORATION OF SOME CULTURAL HERITAGE IN ISFAHAN CITY, IRAN 143

Table 1: Phenotypical identification of isolated bacteria from historical places.

No. of isolates Phenotypic identification Morphology Catalase Oxidase Gram Spore Growth Gelatin Starch Tyrosine on 7% Salt hydrolysis hydrolysis hydrolysis

CH1 Bacillus + + + + + + + + CH2 Bacillus + + + + + + + - CH3 Rod + + - - + - + +

CH: Chehel Sotoun

>gb|CP000485.1| Bacillus thuringiensis str. Al Hakam, complete genome Length=5257091 Score = 658 bits (356), Expect = 0.0 Identities = 358/359 (99%), Gaps = 0/359 (0%) Strand=Plus/Minus 5’CACCTTAGGCGGCTGGCTCCAAAAGGTTACCCCACCGACTTCGGGTGTTACAAACTCTCG CACCTTAGGCGGCTGGCTCCAAAAGGTTACCCCACCGACTTCGGGTGTTACAAACTCTCG TGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCG TGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCG CGATTACTAGCGATTCCAGCTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAA CGATTACTAGCGATTCCAGCTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAA CGGTTTTATGAGATTAGCTCCACCTCGCGGTCTTGCAGCTCTTTGTACCGTCCATTGTAG CGGTTTTATGAGATTAGCTCCACCTCGCGGTCTTGCAGCTCTTTGTACCGTCCATTGTAG CACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCC CACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCC GGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTAAATGATGGCAACTAAAATCAAG GGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTAAATGATGGCAACTAAGATCAAG 3’

Fig. 1: Blast alignment for CH1 isolated from Chehel Sotoun.

gb|JN391533.1| Bacillus licheniformis strain BB10 16S ribosomal RNA gene, partial sequence Length=1472 Score = 664 bits (359), Expect = 0.0 Identities = 359/359 (100%), Gaps = 0/359 (0%) Strand=Plus/Minus 5’CACCTTCGGCGGCTGGCTCCAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGT CACCTTCGGCGGCTGGCTCCAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGT GGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGC GGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGC GATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAAC GATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAAC AGATTTGTGGGATTGGCTTAGCCTCGCGGCTTCGCTGCCCTTTGTTCTGCCCATTGTAGC AGATTTGTGGGATTGGCTTAGCCTCGCGGCTTCGCTGCCCTTTGTTCTGCCCATTGTAGC ACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCG ACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCG GTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGG GTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGG3’

Fig. 2: Blast alignment for CH2 sample isolated from Chehel Sotoun.

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>gb|FJ939560.1| Halomonas sp. HTNK1 16S ribosomal RNA gene, complete sequence Length=1530 Score = 1919 bits (1039), Expect = 0.0 Identities = 1157/1211 (96%), Gaps = 19/1211 (2%) Strand = Plus/Minus 5’ TGGTGTGTGTGGCGACGTGTACAGAGCCCGGAAACCTATACACCGGGACATTCTGATTCA TGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGACATTCTGATTCA CGATTACTAGCGATTCCGACTTCACGGAGTCGAGTTGCAGACTCCGATCCGGACTGAGAC CGATTACTAGCGATTCCGACTTCACGGAGTCGAGTTGCAGACTCCGATCCGGACTGAGAC CGGCTTTTCGGGATTAGCTCACTCTCGCGAGTTGGCAACCCTTTGTACCGGCCATTGTAG CGGCTTTTCGGGATTAGCTGACTCTCGCGAGCTCGCAACCCTTTGTACCGGCCATTGTAG CACGTGTGTAGCCCTACTCGTAAGGGCCATGATGACTTGACGTCGTCCCCACCTTCCTCC CACGTGTGTAGCCCTACTCGTAAGGGCCATGATGACTTGACGTCGTCCCCACCTTCCTCC GGTTTGTCACCGGCAGTCTCCTTAGAGTTCCCACCATTACGTGCTGGCAAATAAGGACAA GGTTTGTCACCGGCAGTCTCCTTAGAGTTCCCGCCATTACGCGCTGGCAAATAAGGACAA GGGTTGCGCTCGTTACGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCAT GGGTTGCGCTCGTTACGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCAT GCAGCACCTGTCTCTGCGTTCCCGAAGGCACCAAGTGATCTCTCACAAGTTCGCAGGATG GCAGCACCTGTCTCTGCGTTCCCGAAGGCACCAAGTGATCTCTCACAAGTTCGCAGGATG TCAAGAGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGT TCAAGAGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGT GCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGA GCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGA CTTATCGCGTTAACTTCGCCACAAAGTGCGCTAGGCACCCAACGGCTGGTCGACATCGTT CTTATCGCGTTAACTTCGCCACAAAGTGCGCTAGGCACCCAACGGCTGGTCGACATCGTT TACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTACCCACGCTTTCGCACCTCAGT TACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTACCCACGCTTTCGCACCTCAGT GTCAGTGTCAGTCCAGAAGGGCGCCTTCGCCACTGGTATTCCTCCCGATCTCTACGCATT GTCAGTGTCAGTCCAGAAGGCCGCCTTCGCCACTGGTATTCCTCCCGATCTCTACGCATT TCACCGCTACACCGGGAATTCTACCTTCCTCTCCTGCACTCTAGCTTGACAGTTCCGGAT TCACCGCTACACCGGGAATTCTACCTTCCTCTCCTGCACTCTAGCTTGACAGTTCCGGAT GCCGTTCCCAGGTTGAGCCCGGGGCTTTCACAACCGGCTTATCAAGCCACCTACGCGCGC GCCGTTCCCAGGTTGAGCCCGGGGCTTTCACAACCGGCTTATCAAGCCACCTACGCGCGC TTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGGAC TTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCAC GGAGTTAGCCGGTGCTTCTTCTGCGAGTGATGTCTTTCCTAATGGGGATTAAACACTAGG GGAGTTAGCCGGTGCTTCTTCTGCGAGTGATGTCTTTCCTAATGGGTATTAACCACTAGG CGTTCTTCCTCGCTGAAAGTGCTT-ACAACCCGAGAGGCCTTCTTTCACACACGCGGCAT GGGCTGGATCAGGCTTGCGCCCATTGTCGATAATTCCCCACTGCTGCTTCCCGGTAGGAG CTGGATCAGGCTTTCGCCCATTGTCCAATATTCCCCACTGCTGCCTCCCG-TAGGAG T-CGG-CC-TGTTCCTCAGTTCCGATGGTGGCGTGATCATCCTCTCAGAACAGCTTACGG TTCGGGCCGTGT-C-TCAGTCCCGATG-TGGC-TGATCATCCTCTCAGACCAGCT-ACGG ATCGTTGCCGTGCTGA-CCATAACCTCACCA-CTAGCTAATCGGACTTAG-CTCTATCTA ATCGTCGCCTTGGTGAGCCATTACCTCACCAACTAGCTAATCCGACATAGGCTC-ATCCA TATAGGCGGGA-ATAG-CGGGA3’

Fig. 3: Blast alignment for CH3 sample isolated from Chehel Sotoun.

Vol. 13, No. 1, 2014  Nature Environment and Pollution Technology BIODETERIORATION OF SOME CULTURAL HERITAGE IN ISFAHAN CITY, IRAN 145

Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Figs. 4-8: Microscopic identification of isolated fungi: Saccharomyces, Alternaria, Cladosporium, Absidia and Penicillium. and then DNA bands were virtualized after staining with In fact, scientists believed that several cryptoendolithic ethidium bromide. At last the PCR products with pure DNA fungi may actively bore into the stone and hence physically bands were sent to Macrogen in South Korea (http:// disrupt its integrity (Gadd 2007). Fungi, unlike the www.macrogen.com) for DNA sequencing. The 16SrRNA phototrophs, do not require light for growth, and so their sequenced data were subjected to BLAST analysis (http:// boring activity can penetrate to greater depths ( Golubic et www.ncbi.nlm.nih.gov/BLAST) for identifying each respec- al. 2005). Indeed, hyphal penetration of materials involves tive 16SrRNA gene amplicon. swelling/deflation effects and channelling of water into the substrate which can form cracks, fissures and crevices, ex- RESULTS AND DISCUSSIONS tend existing ones and lead to the detachment of crystals The results obtained from this study indicated that out of (Suihko et al. 2007). Biochemical actions of fungi as well as 210 samples from different sites gram positive and spore bacteria can lead to microtopological alterations (Gadd forming bacteria were dominant (Table 1). In addition the 2007). They are associated with extracellular mucilaginous Chehel Sotoun were most polluted than the other sites. Fur- substances, which contain, amongst many other metabolites, thermore, Figs. 1-3 confirmed that Bacillus licheniformis acidic, and metal chelating compounds. Acidic metabolites strain BB10, Bacillus thuringiensis str. Al Hakam and (oxalic, acetic, citric, other carbonic acids) deteriorate the Halomonas HTNK1 are the most frequently detected iso- stone minerals by a solubilizing and chelating effect. Or- lates which have been identified using molecular techniques. ganisms of the genus Bacillus have been very frequently iden- In addition, the genus Halomonas HTNK1 was for the first tified on stone buildings (Blazquez et al. 2000, Gaylarde & time isolated from the sites. On the other hand, fungi were Morton 2002, Kiel & Gaylarde 2006). This is not unexpected, isolated from 80 samples. The colonies were directly exam- as they are very common in soil and are able to withstand ined on agar plates and based on macroscopic and micro- extreme environments because of their spore-forming abil- scopic characterization. Five different morphological types ity and ease of culture ( Qi et al. 2011). Kiel & Gaylarde were detected. Fungi genera that were identified in this study, (2006) found that some strains of Bacillus isolates produced were belonged to Genus, Cladosporium sp., Penicillium, Al- acids and surfactants with auto emulsifying activity in the ternaria, Absidia and Saccharomyces. Although the fungi laboratory, indicating that they had the capacity to acceler- were isolated from all the sites, the most polluted area was ate stone degradation. In the present study, isolation of this Chehel Sotoun (Figs. 4-8). genus from most of the parts indicated that the bacteria with The Isfahan city has numerous historical places. These spore could tolerate the arid climate of Isfahan city which in sites are affected by the physical and chemical conditions as further study using HPLC technique could understand the well as the dry climate. The assessed chosen sites are located main mechanisms of the damage. in high traffic routes. Although microorganisms, as a help- ACKNOWLEDGEMENT ing factor, can directly or indirectly contribute to stone deterioration by producing biofilm (Nugari et al. 2009), organic The authors would like to thank Cultural Heritage Organi- and inorganic acids, pigments and several other factors also zation of Isfahan city for the formalities and permitting us cause the damage. The impact of fungi on biodeterioration is to get the samples from the selected sites. due to their physical and chemical activities. In addition, the REFERENCES fungi can cause stone deterioration through various mechanisms such as penetration of hyphae into the stone, produc- Assari, A. and Mahesh, T.M. 2011. Demographic comparative in heritage tion of different pigments and production of organic and in- texture of Isfahan city. Journal of Geography and Regional Planning, organic acids (Shirakawa et al. 2011). Academic Journals, 4(8): 463-470.

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